Raf kinase inhibitor protein enhances neuronal differentiation in human SH-SY5Y cells

Neuronal differentiation has evolved as an essential process even in the adult brain, once disturbed being associated with the pathogenesis of several psychiatric disorders. To study the effects of Raf kinase inhibitor protein (RKIP) on neuronal differentiation, we generated neuroblastoma cell lines overexpressing RKIP (RKIP(+)) and expressing RKIP-directed short hairpin RNA for downregulation of RKIP (RKIP(-)). During a 4-week time course of continuous differentiation by retinoic acid (RA), expression of neuronal and glial markers, intracellular cyclic adenosine monophosphate (cAMP) levels, protein kinase C (PKC) signal transduction to extracellular signal-regulated kinase 1/2 (ERK-1/2) and cellular morphology were investigated in relation to RKIP levels. RKIP(+) cells showed accelerated neurite outgrowth, formation of elaborated neuronal networks and increased neuronal marker expression both in RA-induced differentiation and to some extent even in non-RA-treated cells. RKIP(-) cells showed glial-like cell bodies and increased glial fibrillary acidic protein, suggesting a shift from neuronal to glial phenotype. With respect to differentiation-inducing signal pathways, PKC-mediated ERK-1/2 activation significantly correlated with RKIP levels. Furthermore, basal and forskolin-stimulated intracellular cAMP was potently increased in RKIP(+) cells versus controls. In conclusion, the conserved signal transduction modulator RKIP was shown to enhance several aspects of neuronal differentiation via enhanced crosstalk from PKC to ERK-1/2 and enhancement of G-protein-coupled receptor signaling.